The functional role of the short arm domains of the laminin γ2 chain was investigated by transfecting a series of mutant γ2 cDNAs into keratinocyte cell line LSV5. Cell line LSV5 is derived from the keratinocytes of an H-JEB patient with a homozygous nonsense mutation (R95X) in the gene (LAMC2) coding for the laminin γ2 chain (Miquel et al. 1996
). LSV5 cells do not synthesize the laminin γ2 chain, but express the full repertoire of the laminin chains found in HNKs, including the laminin α3 and β3 chains (Miquel et al. 1996
). Transfection of a wild-type γ2 cDNA restores production of functional α3β3γ2 laminin 5 molecules (Gagnoux-Palacios et al. 1996
). The complementary DNAs coding for mutant laminin γ2 chains were generated by directed mutagenesis of the expression vector pγWT which encodes the full length γ2 polypeptide (Gagnoux-Palacios et al. 1996
). As depicted in B, the mutant cDNA pγNC expresses a recombinant γ2 polypeptide with an internal deletion encompassing the four amino acids (YSGD) within domain III that constitute the proteolytic cleavage site of the γ2 chain (Vailly et al. 1994
; Amano et al. 2000
). In mutant pγGP, the GlyPro residues substitute the YSGD cleavage site and introduce a structural modification from “sheet” to “coil” configuration of the γ2 chain domain III. Mutants pγIII and pγV carry a deletion affecting the egf-like repeat 1 of domain III and repeats 2 and 3 of domain V, respectively. To investigate the functional role of the laminin γ2 chain short arm, we generated a cDNA clone encoding a polypeptide lacking the NH2
-terminal domains IV and V that are excised in the extracellular processing of laminin 5 (plasmid pγC). Further, a polypeptide with an internal deletion encompassing the EGF-like repeat 1 of domain III and the COOH-terminal portion of domain IV (plasmid pγM) was also constructed. Plasmid pγM corresponds to a mutated γ2 chain missing 73 amino acids of the polypeptide sequence detected in a patient suffering from JEB (Pulkkinen et al. 1994
). The deletion was thought to interfere with the extracellular processing of the polypeptide (Amano et al. 2000
The different mutant cDNAs were transiently transfected into actively growing LSV5 keratinocytes. Immunocytochemical analysis of the transfected cultures using pAb SE144 (not shown) and mAb GB3 indicated that all the mutant γ2 polypeptides were actively synthesized and incorporated into laminin 5 heterotrimers (). The ECM deposited on the culture support by cells LγWT, LγNC, LγΔIII, LγΔV, and LγGP transfected with plasmids pγWT, pγNC, pγΔIII, pγΔV, and pγGP was immunoreactive, whereas the ECM deposited by the cell cultures LγC and LγM transfected with plasmids pγC and pγM was not labeled. These results indicate that deletions within the globular domain IV of the γ2 chain prevent secretion and/or deposition of laminin 5 into the ECM, but do not limit laminin 5 synthesis.
Figure 2 Immunofluorescence analysis of laminin 5 synthesis in LSV5 keratinocytes transiently transfected with plasmids encoding the mutant γ2 chains. LSV5 untransfected (a and b) and transfected with pγWT (c and d), pγNC (e and f), pγC (more ...)
To confirm the role of the globular domain of the γ2 polypeptide in deposition of laminin 5 to the cell culture substratum, plasmids pγNC, pγGP, and pγC were stably transfected into LSV5 keratinocytes. Cell lines LγNC and LγGP were generated that are expected to secrete a mutated 440-kD form of laminin 5 with a full length γ2 chain (155 kD), whereas cell line LγC is expected to produce a 400-kD molecule corresponding to laminin 5 with a processed γ2 chain (105 kD). Consistent with the results obtained with transiently transfected LSV5 cells, immunocytochemical analysis of LγNC, LγGP, and LγC cells using pAb SE144 and mAb GB3 detected a strong cytoplasmic labeling in all cell lines. These experiments also confirmed that only the ECM laid down by LγNC and LγGP keratinocytes contains immunoreactive laminin 5 epitopes (not shown). These results indicate that presence of the γ2 short arm is required for deposition of laminin 5 to the culture substratum, but do not rule out the possibility that laminin 5 harboring a γC recombinant chain is secreted into the media without being incorporated into ECM. Expression and secretion of the recombinant γNC, γGP, and γC polypeptides were examined by Western blot analysis of spent medium of LγNC, LγGP, and LγC cells. A unique 105-kD migration band was detected in the LγC medium using pAb SE144, whereas a specific 155-kD band was observed with LγNC and LγGP cells ( A). The intensity of these bands was comparable and was estimated to be threefold weaker than the intensity of the corresponding bands detected in the medium conditioned by wild-type keratinocytes. Thus, although the γ2 short arm is required for incorporation into the ECM, it is not needed for secretion of γ2 chain.
Figure 3 Deposition to the culture substratum of mutant laminin 5 molecules is dependent on the nature of the γ2 chain. (A) Western analysis of spent medium of cultured keratinocytes. 50 μg of proteins from spent medium collected from cultures (more ...)
Incorporation of the recombinant γ2 polypeptides into extracellular laminin 5 molecules was further verified by immunoprecipitation of LγC and LγNC cell culture medium using the mAb K140 and the anti–T7-Tag mAb. As shown in B, comparable amounts of 400- and 440-kD laminin 5 molecules were immunoprecipitated from the LγNC and LγC cell medium, respectively, which attests to the assembly of the 105- and 155-kD recombinant γ2 polypeptides into laminin 5. These data demonstrate that the absence of the γ2 NH2-terminal domains does not hinder the intracellular processing of laminin 5 and its secretion into the culture medium. They also confirm that the tetrapeptide YSGD is the unique physiological cleavage site of the extracellular processing of the γ2 chain.
Artificial epithelia constructed either with HNKs or LSV5 cells expressing a recombinant wild-type γ2 chain have been shown previously to lay down laminin 5 at the interface between the basal cells and the cell culture support (Rosdy et al. 1993
; Gagnoux-Palacios et al. 1996
; Miquel et al. 1996
). Because truncation of the γ2 short arm appeared to prevent deposition of laminin 5 on monolayer submersed cultures, LγC and LγNC keratinocytes were grown to confluence on cellulose acetate filters and exposed to air to obtain stratification into multilayered epithelia. Immunofluorescence analysis of the artificial epithelia using mAb GB3 detected a strong reactivity in the case of LγNC and HKSV cells ( C, b and d), and no reactivity with LSV5 and LγC keratinocytes ( C, a and c). Therefore, these observations confirm that the 400-kD form of laminin 5 produced by LγC keratinocytes is not incorporated into the ECM, and underscore the importance of the γ2 short arm plays in the deposition of laminin 5 at the epithelial–ECM interface.
Because our results suggested that the laminin γ2 short arm is essential for the incorporation of laminin 5 into the ECM secreted by LSV keratinocytes, we verified whether human skin and the matrix secreted by wild-type keratinocytes contain the NH2-terminal γ2 polypeptide generated by the extracellular processing of the 440-kD laminin 5. Specific antiserum pAb SE1097 was generated against recombinant fragment γ50, which is cleaved from the short arm of the γ2 chain ( B). The antibody stained the human epidermal basement membrane in a strong linear fashion ( and ), comparable to the labeling observed with antibody SE144 (not shown). Samples of the ECM produced by cultures of wild-type keratinocytes were then collected from culture dishes and analyzed by immunoblotting using pAb SE144 and pAb SE1097. As shown in C, pAb SE144 recognized the 155- and 105-kD migration bands corresponding to the unprocessed and processed γ2 polypeptide, respectively. pAb SE1097 identified the unprocessed 155-kD γ2 chain and an additional fast-migrating band with the expected mass (50 kD) of the NH2-terminal domains, which are extracellularly excised from the γ2 chain ( D). The finding that only unprocessed γ2 chain is found intracellularly and that the processed γ2 chain and its cleavage fragment are found in the ECM indicate that the 440-kD laminin 5 molecules are proteolytically cleaved into the 400-kD form after incorporation into the ECM. This strengthens the idea that the γ2 short arm plays a role in the integration of laminin 5 in the matrix.
Figure 4 Laminin γ2 short arm drives deposition of laminin 5 to the ECM. Immunofluorescence analysis of control (A) and split skin (B) using pAb SE1097. The strong linear staining indicates that the 440-kD form of laminin 5 and/or the NH2-terminal cleaved (more ...)
To verify this hypothesis, wild-type keratinocytes were transfected with the construct pγ50 encoding the NH2-terminal domains IV and V of γ2 carrying a T7-Tag peptide (). Control cultures were transfected with an expression vector (pβ60) that encodes the NH2-terminal domains III, V, and VI of the laminin β3 chain tagged with the HA epitope (). As shown by immunofluorescence microscopy, mAb T7-Tag reacted with the cytoplasm of the keratinocytes transfected with plasmid pγ50 and labeled the ECM deposited by these cells ( E, a and b). Conversely, the anti-HA mAb stained the cytoplasm of the keratinocytes expressing the recombinant cDNA pβ60, but not the ECM they deposited ( E, c and d). Similar results were obtained by transfection experiments performed using LSV5 cells (not shown). These observations suggest that the short arm of the laminin γ2 chain carries domains essential to the incorporation of laminin 5 into the matrix.
It has been suggested that the globular domain IV of the mouse laminin γ2 chain binds to the ECM protein fibulin 2 (Utani et al. 1997
). However, residue Phe-202 of the γ2 amino acid sequence, which is essential to the interaction, is not conserved in humans. In addition, we were unable to demonstrate interactions between human laminin 5 and fibulin 2 in our experimental conditions (data not shown). Intriguingly, the amino acid sequence of the NH2
-terminal region of the γ2 domain IV is highly conserved in mammals (), which may reflect a relevant physiological role of this portion of the polypeptide. Indeed, it was suggested that disruption of the fibulin 2 binding site could hamper the proteolytic processing of γ2 (Utani et al. 1997
). Therefore, we constructed plasmids pγF1 and pγF2 that encode γ2 polypeptides in which the amino acid sequence SADFSVHKI (residues 199–207) homologous to the active site of the mouse fibulin 2 binding site was partially (pγF1) and totally (pγF2) substituted by alanine residues (). LSV5 keratinocytes transfected with constructs pγF1 and pγF2 were examined by immunofluorescence analysis using mAb GB3. Expression of the mutant laminin 5 molecules resulted in a strong staining of the cytoplasm, and also of the ECM deposited onto the tissue culture support ( A, a and c). Western blot analysis of medium collected from cultures of LγF2 keratinocytes using pAb SE144 detected hybridization bands corresponding to the uncleaved (155 kD) and the proteolytically cleaved (105 kD) pγF2 chain ( B). According to these observations, immunofluorescence examination of frozen sections of artificial epithelia constructed using LγF1 and LγF2 keratinocytes detected a strong reactivity of the basement membrane zone to mAb GB3 ( C, a and b). These results attest to the incorporation of the mutant laminin 5 molecules into the ECM deposited on the cell culture substrate and show that disruption of the region homologous to the putative fibulin 2 binding site of the mouse γ2 short arm does not interfere with the processing and deposition of laminin 5 to the matrix.
Alignment of the Laminin γ2 cDNA Sequences Homologous to the Active Site of Fibulin 2 Binding in the Mouse
Figure 5 Secretion and deposition on the ECM of mutants LγF1 and LγF2 laminin 5 molecules. (A) Immunofluorescence staining of LSV5 keratinocytes transiently transfected with plasmids pγF1 (a and b) and pγF2 (c and d). Double immunofluorescence (more ...)
Reexpression of wild-type laminin 5 restores adhesion of LSV5 cells and H-JEB keratinocytes (Gagnoux-Palacios et al. 1996
; Vailly et al. 1998
). Because the 400-kD molecules of laminin 5 produced by LγC keratinocytes are not deposited into the ECM, LγC cells are expected to retain the poor adhesion capacity of the parental cell line LSV5. Indeed, epidermal sheets of stratified epithelium generated by confluent cultures of LγC, LγGP, and LγF1 keratinocytes spontaneously detached from the culture vessel. In contrast, when LγNC keratinocytes became confluent and stratified, the epidermal sheet firmly adhered to the plastic dish and detachment required enzymatic treatment (not shown). Therefore, attachment of LγC keratinocytes was quantified in detachment kinetic assays in the presence of trypsin/EDTA (Vailly et al. 1998
). The attachment capacity of LγNC keratinocytes was also compared with that of wild-type keratinocytes. Cell suspensions were seeded on petri dishes to obtain exponentially growing cultures. 12 h after plating, the percentage of the adhering LγNC and LγC cells was similar to that of parental LSV5 cells ( A). Although the number of LγNC and LγWT cells resistant to trypsinization increased 48 h after seeding (50% of LγNC cells were dislodged after 14 min), that of LγC and LSV5 cells remained low (50% of dislodged cells after 8 min; B). Therefore, the progressive enhancement of adhesion of LγΝC and LγWT keratinocytes appeared to correlate with accumulation of laminin 5 molecules harboring the γ2 short arm in the matrix. Detachment of LγGP and LγF1 keratinocytes was also assessed in similar experimental conditions. As demonstrated in B, in all these cells other than LγNC synthesis of mutated laminin 5 molecules had no appreciable effect on the strength of cell adhesion.
Figure 6 Detachment assay of keratinocytes expressing mutant laminin 5 and quantification of laminin 5 layered down to the culture substrate. For the detachment assay, cells (2 × 104/cm2) were seeded on a plastic cell culture vessel, left to adhere for (more ...)
To confirm that resistance to trypsinization of LγNC cells correlates with laminin 5 incorporation in the ECM, the amount of laminin 5 layered down by the different γ2 mutant keratinocytes was determined by ELISA assay using mAb GB3. As shown in C, laminin 5 produced by LγNC cells was efficiently deposited on the plastic culture substrate and accumulated with increasing time, whereas mutant γC laminin 5 was inefficiently layered down. LγWT, LγGP, or LγF1 cells layered down comparable amounts of laminin 5. In addition, by seeding the mutant keratinocytes in a plastic vessel coated with different components of the ECM, we assessed the deposition rate of the wild-type and mutant laminin 5 molecules to be independent from the nature of the cell culture substrate on which the keratinocytes are grown (). Plating on a feeder of irradiated mouse 3T3-J2 cells did not modify the deposition pattern of laminin 5 (not shown).
Figure 7 Deposition of mutant laminin 5 is not affected by the cell culture substrate. LSV5 keratinocytes expressing mutant laminin 5 (104 cells per well) were seeded in 96-multiwell plates coated with 10 μg/ml of collagen type I (coll I), collagen type (more ...)
Because the trypsinization assay provides information on the effect of laminin 5 on the strength of cell attachment, the functional role of the laminin γ2 short arm in cell adhesion was further assessed by seeding wild-type primary human keratinocytes on the ECM deposited by the different LSV5 γ2 mutants. The mutant cells were seeded in a range of concentrations leading to deposition of equivalent amounts of laminin 5 48 h after plating. The ECM was prepared after detachment of the cells by EDTA treatment, and concentration of laminin 5 in the ECM was checked by ELISA assays using mAb GB3. Primary wild-type keratinocytes were then seeded and allowed to adhere for 60 min at 37°C. Results show that adhesion of keratinocytes on the matrix secreted by LγNC and LγWT cells was comparable, whereas adhesion on the matrix produced by LγC cells was strongly reduced and equivalent to the values obtained with matrix produced by the γ2-null LSV5 cells (). Adhesion on the matrix produced by the LγF1 and LγGP mutant cells was also reduced to a lesser extent. This result suggests that deposition of these laminin 5 molecules allows interaction of their COOH-terminal G domain with the adhesive integrin receptors at the cell surface, although participation of other cell receptors in adhesion cannot be excluded. However, the cell detachment assay shows that the adhesion strength of mutant LγF1 and LγGP cells is affected, which may reflect a lower binding capacity between laminin 5 secreted by these cells and the culture substrate ( B).
Figure 8 The unprocessed form of laminin 5 promotes cell adhesion. Wild-type keratinocytes (NHK) were seeded on ECM secreted by LSV5 cell expressing laminin 5 harboring the mutant γ2 chains. Cell were left to adhere for 1 h at 37°C. Cell adhesion (more ...)
Because keratinocyte adhesion was measured on a matrix produced by the different LSV mutant cells, we could not determine whether the adhesive effect we observed was directly or indirectly sustained by laminin 5. Nevertheless, our observations demonstrate that the laminin 5 molecules harboring an unprocessed γ2 chain are biologically active and indicate that structural changes in the NH2-terminal region of the globular domain IV affect the biological function of laminin 5.